A liver has 500 or more types of various specific functions. For example, major functions of a liver are plasma protein synthesis and excretion, blood sugar control by gluconeogenesis and glycogen metabolism, lipogenesis, ureogenesis, bile synthesis and excretion, detoxication and so on.
Most substances incorporated into a body are mainly metabolized in a liver. In the field of pharmaceutical development, what type of metabolism pharmaceutical candidate substances will be received in a liver and what type of effect is given to a liver or other organs and tissues are essential data. Further, many chemical substances have been synthesized and discharged into environment up to now. To elucidate what kind of effect these substances have exerted individually or in combination to a human body is socially very important. Toxicity test on liver functions are essential for evaluation of the effects of such chemical substances to a human body.
Mice, rats, rabbits, dogs, monkeys, etc are used at present for safety tests and drug metabolism tests of chemical substances including pharmaceutical candidate substances. Especially in pharmaceutical development, toxicological tests and safety tests using animals are compulsive before entering phase I study for human; therefore, long period and efforts as well as huge costs are required in these tests.
However, there is no guarantee that data obtained by these animal experiments can be applicable to human. In fact, many cases are known wherein a substance not recognized toxicity in animal experiments exhibited toxicity in human or vice versa. Consequently, it is expected up to now that development of many pharmaceutical candidate substances is aborted after entering in phase I study on human, and besides expected that there is many case wherein although a substance has actually no toxicity in human and the development thereof is aborted before entering a clinical trial due to exhibiting strong toxicity in animal experiments.
This may be caused by difference in metabolic functions in a human liver and metabolic functions in livers of mice and rats. Recently, in vitro metabolic tests and toxicity tests using human hepatocytes have been performed. However, amount of livers from brain death patients which were not used for transplantation and amount of human hepatocytes obtained from hepatectomy in tumor excision are far fewer than demanded. Consequently, development of technology for human hepatocyte proliferation is essential for pharmaceutical development.
Necessity of high amount of human hepatocytes is very much alike in an extracorporeal artificial liver. The artificial liver is medical device acting liver function artificially. It is vigorously in progress to develop a hybrid artificial liver combining with the artificial action based on physicochemical principle such as adsorption, dialysis and filtration, along with biological actions using perfusion of an excised liver and liver tissue. In such a development of an artificial liver, it is essential to improve membrane and circuit for enhancing physicochemical function, along with to supply high amount of hepatocytes applicable to human use.
However, in a case of human hepatocytes, it has been considered impossible to serially subcultivate primary cells, which are separated from matured individual. Namely, the matured hepatocytes with adhesion dependency are largely damaged when cells are detached from culture substrate for subcultivation operation and are difficult to re-adhere to culture substrate. Contrary to that, the present inventors of this application have invented the methods for proliferating hepatocytes, wherein small hepatocytes having clonal proliferative ability were isolated from normal hepatocytes separated from human liver, and further, it was carried out to primarily culture the small hepatocytes and then to subculture the cultured hepatocytes; and which were granted patents (JP-A-08-112092, JP No.3266766. U.S. Pat. No. 6,004,810; JP-A-10-179148, JP No.3211941; JP-A-07-274951, JP No. 3157984; and JP-A-09-313172, JP No.3014322).
Although the methods of those patented inventions provide new technique to obtain human hepatocytes in large scale by in vitro proliferation of hepatocytes, there remains a problem of diminishing some liver functions during long period subculture. Therefore, these cells obtained by the above-described methods are useful, for example, for a screening system of medical drug for maintaining liver function, or for a testing system of toxicity and efficacy of medical drugs in terms of certain functions remained after long period subculture, however, these cells are insufficient to use as substitution of human liver function, or for a material of a hybrid-type artificial liver.
As a measure to solve the above problems in proliferation of hepatocytes in vitro, a method for proliferating human hepatocytes in animal body (in vivo) has been proposed.
For example, Heckel, et al. prepared the albumin-urokinase-type plasminogen activator transgenic mouse (uPA-Tg mouse). In this mouse, as the urokinase plasminogen activator (uPA) gene is attached to an enhancer and a promoter of albumin, uPA protein is specifically expressed in a liver (Heckel J L, Sandgren E P, Degen J L, Palmiter R D, and Brinser R L; Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator. Cell 62:447-456, 1990). The color of hepatocytes of said mouse looks white by naked eye due to injury by uPA protein, and some mice die by bleeding or liver failure. It has been known that when normal hepatocytes of lacZ transgenic mouse were transplanted into a spleen of the above-mentioned mouse, the transplanted hepatocytes are attached to the liver and proliferated, and finally the recipient hepatocytes are replaced by the donor hepatocytes (Rhim J A, Sandgren E P, Degen J L, Palmiter R D, and Brinster R L; Replacement of diseased mouse liver by hepatic cell transplantation. Science 263:1149-1152, 1994). Further, Rhim, et al. prepared the uPA-Tg/NUDE mouse by mating between the uPA-Tg mouse and a NUDE mouse which has no T-cell function due to hereditary deletion of thymus. A mouse having rat hepatocytes was prepared by transplantation of rat hepatocytes into the uPA-Tg/NUDE mouse (Rhim J A, Sandgren E P, Palmiter R D and Brinster R L; Complete reconstitution of mouse liver with xenogeneic hepatocytes. Proc. Natl. Acad. Sci. USA 92:4942-4946, 1995). However, there is no report on a chimeric mouse carrying human hepatocytes using said mouse.
Dandri, et al. prepared the uPA-Tg/Rag2 mouse by mating the uPA-Tg mouse and Rag2 mouse which is a knockout mouse having an immunodeficient characteristics. It has been reported that 15% of the mouse liver was replaced by human hepatocytes when human hepatocytes obtained from a human liver were transplanted into a liver of the uPA-Tg(+/−)/Rag2 mouse. Further they were succeeded in in vivo infection with hepatitis B virus to said mouse (Dandri M, Burda M R, Torok B, Pollok J M, Iwanska A, Sommer G, Rogiers X, Rogler C E, Gupta S, Will H, Greten H, and Petersen J; Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Hepatology 33:981-988, 2001).
Also, the inventors of this present invention have disclosed in a prior patent application (JP-A-2002-45087) that in a chimeric mouse produced by transplantation of human hepatocytes into an uPA-Tg/SCID mouse obtained by mating the uPA-Tg mouse and SCID mouse which is an immunodeficient mouse, the transplanted human hepatocytes have substantially taken place functions of a mouse liver. In this chimeric mouse of the prior invention, mouse hepatocytes have been dysfunctioned due to expression of uPA gene, and thus the liver function is maintained by the transplanted human hepatocytes. Therefore, said chimeric mouse is quite useful as an experimental animal for evaluating toxicity and efficacy of test substances because the in vivo function of transplanted human hepatocytes may be evaluated precisely. However, the chimeric mouse of the prior invention cannot live 50 days or longer after transplantation of human hepatocytes. Also, due to proliferation normalized mouse hepatocytes in the course of growing, proliferation efficiency of transplanted human hepatocytes was low and replacement ratio of human hepatocytes remained about 50%.
Above finding has also been supported by a recent report published by Mercer, et al. (Mercer D F, Schiller D E, Eliiott J F, Douglus D F, Hao C, Ricnfret A, Addison W R, Fischer K P, Churchill T A, Lakey J R T, Tyrrell D L J and Keteman N M; Hepatitis C virus replication in mice with chimeric human livers. Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Nature Medicine 7:927-933, 2001). Mercer, et al. have reported that the uPA-Tg/SCID mouse, prepared by similar procedures as described by the inventors of this present application, was transplanted with thawed human hepatocytes which had been stored in a frozen state; as the results, less than 2 mg/ml of human albumin was detected in mouse serum (equivalent to approximately less than about 1 mg/ml in blood) implying that about 50% of liver have been replaced by human hepatocytes.
As described above, a chimeric mouse which is prepared by a transplantation of human hepatocytes into an immunodeficient hepatopathy mouse (uPA-Tg/ SCID mouse) was insufficient as means to proliferate transplanted human hepatocytes in large scale in a mouse, even though a chimeric mouse itself had usefulness (for example, for in vivo testing of toxicity or efficacy to human hepatocytes).
Moreover, the chimeric mouse transplanted with human hepatocytes can not live for long period of time; and since mouse hepatocytes proliferate in a course of its growing, availability of such a chimeric mouse as an in vivo evaluation system of toxicity or efficacy against human hepatocytes has been limited.
Scope of the present invention is to provide, as a method for proliferating human hepatocytes, an improved method for proliferating human hepatocytes sufficiently in a mouse body.
Also, to provide a method for separation and recovery of human hepatocytes propagated in a mouse body by extension of life time is also included in the scope of the present invention.
Further, the scope includes to provide a method for obtaining a large number of chimeric mice carrying human hepatocytes with certain specifications by means of transplantation of separated human hepatocytes into plural mice and proliferation in mice bodies, and a method for obtaining human hepatocytes with certain specifications separated from said mouse in large scale.
Furthermore, to provide a method for application of separated human hepatocytes is also included in the scope.
In addition, the scope of the present application is to provide a useful monoclonal antibody useful to operate the above-described each invention, and a new hybridoma cell line producing said monoclonal antibody.